3D-3-culture: A tool to unveil macrophage plasticity in the tumour microenvironment

Abstract

The tumour microenvironment (TME) shapes disease progression and influences therapeutic response. Most aggressive solid tumours have high levels of myeloid cell infiltration, namely tumour associated macrophages (TAM). Recapitulation of the interaction between the different cellular players of the TME, along with the extracellular matrix (ECM), is critical for understanding the mechanisms underlying disease progression. This particularly holds true for prediction of therapeutic response(s) to standard therapies and interrogation of efficacy of TME-targeting agents. In this work, we explored a culture platform based on alginate microencapsulation and stirred culture systems to develop the 3D-3-culture, which entails the co-culture of tumour cell spheroids of non-small cell lung carcinoma (NSCLC), cancer associated fibroblasts (CAF) and monocytes. We demonstrate that the 3D-3-culture recreates an invasive and immunosuppressive TME, with accumulation of cytokines/chemokines (IL4, IL10, IL13, CCL22, CCL24, CXCL1), ECM elements (collagen type I, IV and fibronectin) and matrix metalloproteinases (MMP1/9), supporting cell migration and promoting cell-cell interactions within the alginate microcapsules. Importantly, we show that both the monocytic cell line THP-1 and peripheral blood-derived monocytes infiltrate the tumour tissue and transpolarize into an M2-like macrophage phenotype expressing CD68, CD163 and CD206, resembling the TAM phenotype in NSCLC. The 3D-3-culture was challenged with chemo- and immunotherapeutic agents and the response to therapy was assessed in each cellular component. Specifically, the macrophage phenotype was modulated upon treatment with the CSF1R inhibitor BLZ945, resulting in a decrease of the M2-like macrophages. In conclusion, the crosstalk between the ECM and tumour, stromal and immune cells in microencapsulated 3D-3-culture promotes the activation of monocytes into TAM, mimicking aggressive tumour stages. The 3D-3-culture constitutes a novel tool to study tumour-immune interaction and macrophage plasticity in response to external stimuli, such as chemotherapeutic and immunomodulatory drugs.

Monocytes as Endothelial Progenitor Cells (EPCs), Another Brick in the Wall to Disentangle Tumor Angiogenesis

“…To unravel the role of monocytes as EPCs in cancer context, we employed a 3D co-culture system [44] of human breast cancer cells (HCC1954) and human monocytes. In co-culture, hvWF positive cells also expressed FN ( Figure 5A), which suggested that monocytes are differentiating into ECs.…”

A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids

“…However, the cell death response was decreased to around 30% in day 10 spheroids. The decrease in ferroptosis could be caused by an increase in complexity of spheroids over time and secretion of extracellular matrix [16]. For day 1 SKOV spheroids, approximately 85% of cell death was observed after stimulation with ML-162.…”

“…Therefore, with regard to the responses to therapy, including to cell death, 3D structures are better test models for drug screening compared to 2D monolayers [13].Spheroids are one of the most widely used 3D models and can be formed by seeding cells in nonadherent plates allowing for self-assembly ( Figure 1A) [7]. In spheroids, the ECM can be produced by cells, which acts as an extra barrier for drugs to cross in order to induce tumour cell death [14][15][16]. Moreover, the compactness and stiffness of spheroids resemble the in vivo hypoxic tumour microenvironment [17], which can cause drug resistance through cell cycle arrest and downregulation of pro-cell death proteins (e.g., caspase-3 in apoptosis) [18].…”

Designer Biomaterials to Model Cancer Cell Invasion In Vitro: Predictive Tools or Just Pretty Pictures?

“…TAMs migrate to the cancer microenvironment as monocytes and are differentiated and polarized at the tumor site, typically to an alternatively activated, or M2, phenotype with angiogenic and ECM remodeling characteristics 57–59. The presence of TAMs are associated with poor prognosis as confirmed for a wide range of solid tumors using histology 59. However, histology alone is unable to capture the dynamic processes of TAM recruitment to the microenvironment and cancer cell invasion following TAM accumulation.…”

“…However, histology alone is unable to capture the dynamic processes of TAM recruitment to the microenvironment and cancer cell invasion following TAM accumulation. Thus, hydrogel disease models incorporating TAMs aim to recapitulate aggressive tumor microenvironments 59–62…”